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3M Environmental Laboratory FinalReport-AnalyticaSltudy Single-DoseIntravenousPharmacokineticStudy ofT-6053 inRabbits In-VivoStudyReferenceNumber: HWI#6329-136 Study Number: AMDT-010495.1 TestSubstance:FC-99 (T-6053) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,NIN 55106 Name and Address ofTestingFacility: 3M EnvironmentalTechnology& Services 935 Bush Avenue St.Paul,MN 55106 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal ExtractioonfFluorideby Means of a Modified Dohnnann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means ofan Orion EA940 Expandable Ion Analyzer ExtractionofFluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochemicalsUsing ElectrospmyMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectivEelectrode InitiatioDnate: See attachedprotocol Author:James D. Johnson Approved By: SaatmmuedesyDs@ tolson Study ir tor Vompletion Date OOOCOI- I 1.0 SUMMARY Rabbiltivewrereanalyzebdycombustiofnorfluoricnoentenatt48 hoursafter individuarlabbitswere dosed intravenouslwyitha rangeofsingledosesof FC-99 (onerabbit/dosgeroup).The fluorinweas detectedaftera singledose of 20 ug/kg,but not aftera 0.8,4, 10 ug/kg dose at48 hourspostdose.Doses are calculatewdithrespectto theC8 solids. The perfluorooctanesulfonsaatletform of FC-99 does notchange theavailabiliotfy totalorganicfluorineinwhole liverafterintravenouisnjectionT.he analysisof liverfortotalorganicfluorinweillprovidea convenientmarker toassessthedermal absorptionof FC-99. 2.0 INTRODUCTION Thisstudywas designedtoprovideinformationastowhetherthe perfluorooctanesulfonataeniongoes totheliverand othertissueasftera dose of T-6053 (a 0.04% solutionofFC-99 solids)isadministeredinan intravenousdose;and to ascertaitnhechange inconcentratiownith time afterdose inserum and liver.FC-99 isa solutionofperfluoroalkylsulfonatpeesr:flourooctanesulfon(ataebout20%), C4 - C7 perfluoroalkylsulfona(taebsout5%), and water(75%). The sulfonateasre as the diethanolaminesalt. Itisknown from studiesdone previouslywithratst,hatthehalf-lifoefperfluorooctanesulfonataenionisquitelong(>I month). Itwas shown ina previousstudy thatthehalf-lifoefperfluorooctanesulfoniast>e1 month inrabbitsafteran intravenoudsose (HWI#6329-159). Itisnotexpectedthatthehalf-lifienrabbits would be differenwtithjustthesaltform oftheperfluorooctanesulfonactheanged, as itiscomparingFC-95 (potassiumsaltw)ithFC-99. Itwas notedinthepreviousintravenoustudywiththeperfluorooctanesulfonattheat theserum levelslaggedand even decreasedaftertheinjectioannd thenreturnedto a maximum at48 hours. Itwas clearfrom the28 day study(HWI#6329-159) thattheperfluorooctanesulfonatewould providea marker fordermal studiesforwhich therewas reasonable expectatiotnhatperfluorooctanesulfonawtoeuld be formed asa biotransformation product.Itwillthereforealsobe a usefulmarker fortheassessmentofthedermal absorptionof FC-99. 0 0 0 00 2 3.0TEST MATERIALS 3.1Test,Controla,nd ReferenceSubstanceasnd Matrices 3.1.1AnalyticaRleferenceSubstance:FC-95,lot161or 171.They are equivalent. 3.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 3.1.3AnalyticalControl Substance: None 3.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 3.2 Source of Materials: 3M ICP/PCP DivisionforFC-95, bovine liverfrom grocerystore,bovine serum from Sigma Chemical Company 3.3.Purity and Strength of Reference Substance: Responsibilitoyf Sponsor. 3.4 Stabilityof Reference Substance: To be determinedby Sponsor. 3.5Storage Conditions for Test Materials:Room temperatureforFC-95. For biologicalsamples the storageis-20+10' C. 3.6 Dispositionof Specimens: Biologicaltissuesand fluidswillbe retainedper GLP Regulationforthetime periodrequiredforstudieslongerthan 28 days. 4.0 EX]PERIMENTAL -Overview Serum and tissuesfrom animalsdosed as described(HWI#6329-136) were availableforanalysisforfluorinecompounds. Sinceperfluorooctanesulfonate anion isnot biotransformed,the analysiswas accomplished with combustion and subsequentanalysisforfluorine.The fluorinedataarerelateddirectlyto perfluorooctanesulfonactoencentrationT.he fluorineanalysisof serum collectedat differentime intervalasfterdosingprovidesdatawhich can be interpreted pharmacokineticallyT.he dataare toprovideevidencethatperfluorooctanesulfonate and/ortotalorganicfluorinein livercan be used to assessdermal absorptionof FC-99. 5.0 EXPERIMENT L -METHODS 5.1 AMDT-M-1-0, Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 Organic HalideAnalyzer-Liver 5.2 AMDT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 0()000@,'i 3 5.3AMDT-M4-0, ExtractiofnFluorochemicaflrsomRabbitLiver 5.4AMDT-M-5-0,AnalysiosfRabbiLtiverExtracftorFluorochemicUaslisng ElectrospmyMass Spectrometry 5.5AMDT-M-8-0. Analysisof FluorideUsing theSkalarSegmented Flow Analyzerwith Ion SelectivEelectrode 6.0 DATA ANALYSIS The datafrom combustionanalysisof liverfortotalorganicfluorineareattached. The liverat48 hourspostdose indicatetshatonlythe250 mg/kg dose of T-6053 resulteidnsignificatnottalorganicfluorinientheliver(35ug intotaliver)T.hisis a 0.04% solutionofan aqueoussolutioninwhich theperfluorooctanesulfonastaeltis about 20%. Thiscorrespondsto a dose intermsof perfluorooctanesulfonastaeltas about0.02mg/kg. (The controlvalueisestimatedtobe about 15 ug totalinthe whole liverb,utthevalueisbelow thequantitatiolnimitand isextrapolatefdrom below thecalibrationftheinstrument.T)hisresultisasexpectedfrom a previous studyinrabbitswhen perfluorooctanesulfonatsethepotassiumsaltwas injecteadnd onlydosesabove 0.6mg/kg resultedinsignificanatmounts oftotalorganicfluorine intheliver.There isenough oftheperfluorooctanesulfonpartesenttoindicattehat ifappreciablaemounts ofFC-99 areabsorbedina dermal absorptionstudy,itwill be observed. Other datawas collectedusingelectrospramyass spectrometryand Skalarsegmented flow analyzerwithion selectiveelectrod(eseeappendices)T.hisdata,although supportivei,ntheopinionoftheStudy Directorisnot requiredtoreachthe conclusionstatedhere and thereforeisnot discussedindetail. 6.1 Circumstances that May Affectthe Quality of the Data: The problem with thisanalysisisthatthereistoo shorta timeperiodused forthestudy.Inother studiesi,thasbeen observedthatthereisa laginserum concentrationusntil 48 hours. There didn'tappear to be any advantage in doing serums forthisstudy, sincethetimeperiodwas too shortand inview oftheresultsfrom study HWI#6329-159. 0()000.1 4 7.0CONCLUSION Theperfluorooctanesulsfaolfntoartme(diethanolamoifnFeC)-99doesnotchange theavailabiliotfytotalorganicfluorineinwhole liverafterintravenousinjection. The analysisofliverfortotalorganicfluorinweillprovidea convenientmarker to assessthedermal absorptionofFC-99. However a largerdose willbe required. 8.0 MAINTENANCE OF RAW DATA AND RECORDS 8.1 Raw Data and Data: Raw data,approved protocola,pproved finalreport, appropriatsepecimens,and electronidcatawillbe maintainedintheAMDT archives. 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1Protocoland FinalReport:HWI#6329-136 "Single-DoseIntravenous PharmacokineticStudyofT-6053 inRabbits"(ProtocoltypeTP8084.PK for dosing of animals,tissuecollectione,tc.) 9.1.2 AnalyticaplrotocolAMDT-0 10495.1 9.2 Signed Reports from IndividualScientistsN:one 9.3 QualityAssurance Unit Statement:See attached 9.4 Key PersonnelInvolvedinthe Study: See attached 9.5 Materialsand Equipment: See methods 9.6 Solutions,Reagents,and Standards:See methods 9.7 Sample Preparation:See methods 9.8 QualityControlPractices:See methods 9.9 TestMethods: See ProtocolAMDT-010495.1 9.10 Instrument Settings:See methods 0()()001,35 9.11Data:Seeattached. 9.11.1Summary and raw data;ug F-inwhole liverasdeterminedby thermal extractiofnollowedby analysisusingOrion ionanalyzer. 9.11.2Summary and raw data;analysisof liverextractussingelectrospray mass spectrometry. 9.11.3Sunnnary and raw data;ug F-inwhole liveras determinedby thermal extractiofnollowedby analysisusingSkalarsegmented flow analyzerwith ion selectivelectrode. 6 9.1.1Protocoland FinalReport:HWI#6329-136 "Single-DoseIntravenousPhannacokineticStudy of T-6053 in Rabbits"(ProtocoltypeTP8084.PK for dosingof animals,tissuecollectione,tc.) 0()Ooo-i HAZLCECCN W IS C 0 N S IN POST OFFICE BOX 7545 MAF)ISON, V\/l 53707-7545 Sponsor: 3M St. Paul, Minnesota .1CORNING Cornpiiiy FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits Author: Steven M. Glaza Study Completion Date: March 31, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryPro.iectIdentification: HWI 6329-136 Page 1 of 25 P h o n e 6 0 8 -2 4 1 -4 4 7 1 E X P R E S S -M A IL D E L IV E R Y . 3301 KINSMAt4 BLVD 1 Fa x MADISO@J, 0()()O()LI 6 0 8 -2 4 1 -7 2 2 7 wi 53704 Page 2 of 25 HWI 6329-136 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordance with the Food and Drug Administration (FDA) Good LaboratoryPractice Regulations,21 CFR 58.35 (b) (6) (7). The following inspections were conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Management 12/09/94 12/09/94 Protocol Review 12/27/94 12/27/94 Sample Collection 02/15/95 02/15/95 Data/Report Review 03/30/95 03/30/95 Report Rereview 12/09/94 12/27/94 02/15/95 03/30/95 01/10/95 01/10/95 03/10/95 04/10/95 e@ecciill'Dii@/aaanM. Date Representative Quality Assurance Unit 0()()00!) Page 3 of 25 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits HWI 6329-136 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental/In-lifeStart Date Experimental/In-lifeTermination Date T-6053 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, Wl 53704 December 27, 1994 December 29, 1994 Page 4 of 25 HWI 6329-136 KEY PERSONNEL Acute Toxicology Steven M. Glaza Study Director Manager Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-lifeSupervisor Rose M. Bridge Report Supervisor Ouality Assurance Anatomical Pathology Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Sherry R. W. Petsel Manager 0()OOIIL Page 5 of 25 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and ControlMaterials Test System Procedures Results Discussion Signature Table I IndividualBody Weights(g) 2 Individual Clinical Signs Appendix A ProtocolDeviations, Protocol TP8084.PK HWI 6329-136 Paqe 2 3 4 6 7 7 7 8 9 11 11 11 12 13 14 15 16 Page 6 of 25 HWI 6329-136 SUMMARY This study was done to assess the level of systemic exposure of T-6053 when administeredby intravenousinjectionto rabbits. Female Hra:(NZW)SPFrabbits were assigned at random to five groups (one/group). On Day 0, the animalsreceiveda single intravenousinjectionof the vehicle(sterilewater for injection)or 10, 50, 125, or 250 mg of T-6053/kgof body weight (Groups 1 through 5, respectively). The dose volume was 0.5 mL/kg for all groups. Clinicalobservationswere conducted at approximately0.5, 2, 4, 24, and 48 hours after intravenousinjection. Body weightswere determinedjust before test material administration(Day 0). A blood sample (approximately 4 mL) was collectedfrom an auricularartery or marginalear vein of the animalsat 2-, 4-, 6-, 8-, 12-, and 24-hourspost-injection.In addition,at the time of in-lifetermination(48-hourspost-injection)a,pproximately20 mL of blood was obtained from each animal. All samples were centrifuged, separatedinto serum and cellularfractions,and sent to the Sponsor. Approximately48 hours post-injection,the animalswere anesthetizedwith sodium pentobarbital,bled via the posteriorvena cava, and exsanguinated. An abbreviated gross necropsy examinationwas not done, however, tissues were collected. The whole liver, bile, and both kidneysfrom each animal were collectedand sent frozento the Sponsorafter terminationof the in-life phase. All five animals appearednormal throughoutthe study. 00001,'i Page 7 of 25 HWI 6329-136 OBJECTIVE The objectiveof this study was to assess the level of systemicexposure to the test material,T-6053,when administeredas a single intravenousinjection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordancewith the U.S. Food and Drug Administration'sGood LaboratoryPracticeRegulationsfor Nonclinical LaboratoryStudies,21 CFR 58, with the exceptionthat analysisof the test mixtures for concentration,homogeneity/solubilitya,nd stabilitywas not conducted. All proceduresused in this study were in compliancewith the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director,the study did not unnecessarilyduplicateany previouswork. TEST AND CONTROL MATERIALS Identification The test material was identifiedas T-6053 and describedas a clear, colorless liquid. The controlmaterialwas SterileWater for Injection,USP (Abbott Laboratories, Lot No. 86-748-DM-02;Exp. March 1, 1996), and was described as a clear,colorlessliquid. Purity and Stability The Sponsor assumes responsibilityfor test materialpurity and stability determinations(includingunder test conditions). A sampleof the test material/vehiclemixturesfor concentration,solubility,homogeneity,and stabilityanalyseswas not taken before administrationas this was not requestedby the Sponsor. The purity and stabilityof the USP grade control material were consideredto be adequatefor the purposesof this study. Storage and Retention The test material was stored at room temperature. The control materialwas stored refrigerated. Any unusedtest materialwas returnedto the Sponsor after completionof all in-lifetestingaccordingto HazletonWisconsin(HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testingand will not be discardedafter issuanceof the final report. 00001,1 Page 8 of 25 HWI 6329-136 Safety Precautions The test and control material handling procedures were accordingto HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPFstrain were receivedfrom HRP, Inc., Kalamazoo, Michigan on November 30, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt,the animalswere acclimatedfor a period of at least 7 days. During acclimationand throughoutthe study, the animalswere individually housed in screen-bottomstainlesssteel cages in temperature-and humiditycontrolledquarters. Environmentalcontrols for the animal room were set to maintain a temperatureof 190 to 230C, a relative humidity of 50% 20%, and a 12-hourlight/12-hourdark lightingcycle. In cases where variationsfrom these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of LaboratoryRabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturerfor nutritionalcomponentsand environmental contaminants. Samples of the water are periodicallyanalyzed by HWI. There were no known contaminantsin the feed or water at levels that would have interferedwith or affectedthe resultsof the study. Selection of Test Animals The animals were identifiedby animal number and correspondingear tag and were selected at random based on health and body weight requirements. ()()OOIIJ Page 9 of 25 HWI 6329-136 Study Design Female animalsweighing from 2,572 to 2,836 g at initiationof treatmentwere placed into the followingstudy groups: Group Dose Level Dose Volume Number Treatment (mg T-6053/kq) (mL/kq) of Animals I (Control) 0 2 T-6053 10 3 T-6053 50 4 T-6053 125 5 T-6053 250 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 SterileWater for Injection,USP. Justificationfor Species Selection Historically,the New ZealandWhite albino rabbit has been the animal of choice because of the large amount of backgroundinformationon this species. PROCEDURES Dose Preparation and Administration The test materialwas dilutedwith SterileWater for Injectionto achieve a specificconcentrationfor each dose level. An individualdose of each respectivetest solutionor controlwas calculatedfor each animal based on its body weight on the day of treatment. The respectivetest solutionwas administeredby intravenousinjectioninto a marginalear vein. The dose was given as a slow push (approximately30 secondsto 2.5 minutes in duration). The prepared test solutionswere stored at room temperatureuntil administered. After administration,any remainingtest solutionswere discarded. Reason for Route of Administration Intravenousinjection is an acceptableroute to assess systemic exposure. Observations of Animals Clinical observationswere conductedat approximately0.5, 2, 4, 24, and 48 hours after intravenousinjection. Body weights were determinedjust before test materialadministration(Day 0). Page 10 of 25 HWI 6329-136 Sample Collection A blood sample (approximately4 mL) was collectedfrom either ear via the catheterizationof the auriculararteryor from the marginalear vein of all animalsat 2, 4, 6, 8, 12, and 24 hourspost-injection.At the time of necropsy (approximately48-hourspost-injection),approximately20 mL of blood was obtainedfrom the posteriorvena cava of each animal. All sampleswere stored at room temperatureuntil centrifuged and separated into serum and cellularfractions. The blood sampleswere then stored in a freezerset to maintain a temperatureof -200C 100C until shipped to the Sponsor. Pathology At terminationof the in-lifephase (approximately48-hourspost-injection), animals were anesthetizedwith sodium pentobarbital,bled via the posterior vena cava, and exsanguinated. An abbreviatedgross necropsy examinationwas not conducted,however,tissueswere collected. The whole liver, bile, and both kidneys from each animal were collected and immediatelyplaced on dry ice, then placed in a freezerset to maintain a temperatureof -200C 100C.' After tissue/bilecollection,the animalswere discarded. Shipment of Tissues After completionof the in-lifephase the blood samples,livers,bile, and kidneys were sent frozen (on dry ice) to the Sponsor (JamesD. Johnson,3M E.E. & P.C., Bldg. 2-3E-09,935 Bush Avenue, St. Paul, MN, 55106). The Sponsor is responsiblefor the retentionand dispositionof the samples. HWI does not accept any responsibilityfor the analysisof the samples collectedin this study nor are these resultspresentedin this report. StatisticalAnalyses No statisticalanalyseswere requiredby the protocol. Location of Raw Data, Records, and Final Report The raw data, records,and an originalsigned copy of the final report will be retained in the archives of HWI in accordancewith HWI SOP. 0()0011-fl Page 11 of 25 RESULTS Body Weights Individualbody weights at initiationare in Table 1. HWI 6329-136 Clinical Observations Individualclinical signs are in Table 2. All five animals appeared normal throughout the study. Pathology All animals survived to termination of the in-life phase and were not examined grossly when sacrificed. DISCUSSION The level of systemic exposure of T-6053 was evaluated in female albino rabbits when administeredas a single intravenous injection at levels of 0, 10, 50, 125, and 250 mg/kg. All animals appeared normal throughout the study following administrationof this material. SIGNATURE Steven M. Glaza Study Director Acute Toxicology 3 -3 1-c@6- Date 0()001,tl Group 1 2 3 4 5 Page 12 of 25 Table 1 IndividualBody Weights (g) Dose Level (mg/kq) Sex Animal Number 0 Female F52856 10 Female F52857 50 Female F52861 125 Female F52862 250 Female F52863 HWI 6329-136 Initial 2,833 2,802 2,572 2,746 2,836 Dose Level Group (mg/kq) Sex 1 0 Female Page 13 of 25 Table 2 IndividualClinicalSigns Animal Number Observation 0.5 F52856 Appeared normal HWI 6329-136 Hour 2 4 24 48 1( 2 10 Female F52857 Appeared normal 1( 3 50 Female F52861 Appeared normal 4 125 Female F52862 Appeared normal 1( 5 250 Female F52863 Appeared normal Condition existed. 0 Page 14 of 25 APPENDIX A Protocol Deviations Protocol TP8084.PK HWI 6329-136 0()()O'A-."Ol Page 15 of 25 Protocol Deviations HWI 6329-136 Protocol Page 8, 7. ExperimentalDesign, C. Dosing Procedures,(1) Dosing Route. Intravenous injectioninto a marginalear vein over approximately30 to 60 seconds. Actual Procedure - Animal Nos. F52856 (Group 1), F52861 (Group 3), and F52862 (Group4), had a dosing duration of 157, 91, and 99 seconds, respectively. These deviationsare not consideredto have had an adverse effect on the outcome of the study. HAZLEECC:N W IS C 0 N S IN POST OFF ICF BOX 7F)45 MADISON. Wi 53 107 71),15 Page 16 of 25 a CORIYING C(xyil)any Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Phamacokinetic Study of T-6053 in Rabbits Date: December 13, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProject Identification: HWI 6329-136 P ho n e EXPRESS 6 0 8 2 4 1 4,1 1 1 MAIL DF.L.IVERY 3301 K IN S M A N BLVD Fax MADISON 608 wi 2 4 1 1221 53 704 0 0 O'A-4:13 Page 17 of 25 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-136 T-6053 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 27, 1994 December 29, 1994 February 2, 1995 Page 18 of 25 TP8084.PK Page 3 1. Study Single-DoseIntravenousPhamacokinetic Study in Rabbits 2. Purpose To assessthe level of systemicexposurewhen the test material is administeredas a single intravenousinjectionto rabbits 3. Regulatory Compliance This studywill be conductedin accordancewith the followingGood LaboratoryPractice Regulations/Standards/Guidelinweisth the exceptionthat analysisof the test materialmixturesfor concentrations,olubility,homogeneity,and stabilitywill not be conducted: C ] Conduct as a Nonregulated Study [XI 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(81)30(Final)(OECD) 59 Nohsan No. 3850 (Japanese MAFF) NotificationNo. 313 (JapaneseMOHW) All proceduresin this protocolare in compliancewith the Animal WelfareAct Regulations. In the opinionof the Sponsorand study director,the study does not unnecessarilyduplicateany previous work. 4. Ouality Assurance The protocol,studyconduct,and the finalreportwill be auditedby the QualityAssuranceUnit in accordancewith HazletonWisconsin (HWI) StandardOperatingProcedures(SOPS)and policies. 5. Test Material A. Identification T-6053 B. PhysicalDescription (To be documentedin the raw data) C. Purityand Stability.. The Sponsorassume;responsibilitfyor purityand stability determinations(includingunder test conditions).Samplesof test material/vehiclmeixture(s)for concentrations,olubility, homogeneity,and stabilityanalyseswill be taken before administrationif requestedby the Sponsor. These samples(if taken)will be sent to the Sponsorafterexperimental terminationfor possibleanalysis. Page 19 of 25 TP8084.PK Page 4 D. Storacie Room temperature E. Reserve Samgles Reservesampleswill not be requiredfor this study. F. Retention Any unusedtest materialwill be returnedto the Sponsorafter completionof the in-lifephase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. ControlMaterial A. Identification Sterilewater for injection B. PhysicalDescription Clear,colorlessliquid C. Purityand Stability The purityand stabilityof this USP grade materialis consideredto be adequatefor the purposesof this study. D. Storage Refrigerated E. Reserve Samgles See Section,5. E. ReserveSamples F. Retention Any remainingcontrolmaterialmay be used for other testing and will not be discardedafter issuanceof the final report. G. Safety Precautions As required by HWI SOPs and policies 7. Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP,Inc. (3)Age at Initiation Adult Page 20 of 25 TP8084.PK Page 5 (4) Weightat Initiation 2.5 to 3.5 kg (5)Number and Sex 5 females (6) Identification Individualnumberedear tag (7) Husbandry (a) Housing Individually,in screen-bo.ttosmtainlesssteel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMIFeeds,Inc.). The food is routinelyanalyzedby the manufacturerfor nutritionalcomponentsand environmentalcontaminants. (c)Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness,and specifiedmicrobiologicalcontentand for selectedelements,heavy metals, organophosphates,and chlorinatedhydrocarbons. (d)Contaminants There are no known contaminantsin the food or water thatwould interferewith this study. (e) Environment Environmentalcontrolsfor the animalroom will be set to maintaina temperatureof 19*C to 23*C, a relativehumidity of 50% +20%, and a 12-hour light/12-hourdark cycle. (f) Acclimation At least 7 days (8)Selectionof Test Animals Based on health and body weight accordingto HWI SOPS. An adequate number of extra animalswill be purchased so that no animal in obviouslypoor health is placedon test. (9)Justificationfor SpeciesSelection Historically,the New ZealandWhite albinorabbit has been the animal of choice becauseof the large amount of backgroundinformationon this species. 0 Page 21 of 25 TP8084.PK Page 6 B. Dose Administration (1) Test Groups fi[2-UR 1 2 3 4 5 Dose Level -mg/kg)" 0 (Control) 10 50 125 250 Number of Females 1 1 1 1 1 a The dose volume will be 0.5 mL/kg of body weight. C. Dosing Procedures (1) Dosing Route Intravenousinjectioninto a marginalear vein over approximately30 to 60 seconds. (2) Reason for Dosi@q Route Intravenousinjectionis an acceptableroute to assess systemic exposure. (3)Dosing Duration Single dose (4) Dose Preparation The testmaterialwill be dilutedwith sterilewater for injectionto achievea specificconcentrationfor each dose level. Individualdoses will be calculatedbased on the animal'sbody weight taken just beforetest material administration.The preparedtest mixtureswill be stored at room temperatureuntil administration. D. Observationof Animals (1) ClinicalObservations The animalswill be observedfor clinicalsigns of toxicityat approximatel0y.5,2.02 4.0124, and 48 hours after treatment. (2) Body Weights Just beforetest materialadministration. (3)Samgle Collections (a) Frequenc 2, 4, 6, 8, 12, 24, and 48 hours post-injection 0 O'A.-Wo Page 22 of 25 TP8084.PK Page 7 (b) Number of Animals All (c) Method of Collection Blood samples (approximately4 mL) will be collected from either ear via the catheterizationof the auricularartery or from the marginal ear vein at 2, 4, 6, 8, 12, and 24 hours post-injection. Approximately20 mL of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava of each animal at the time of necropsy (48 hourspost-injection). Approximately 20 mL of blood will be collected from moribund animalsduring the study, also, if possible. The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractionsstored in a freezer set to maintain a temperature of -20*C TIO'C. The separated serum and cellularfractionswill be sent frozen on dry ice to the Sponsor after experimental termination. Samples will be shipped to: James 0. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or his alternatewill be notifiedby telephoneat (612) 778-5294 prior to the shipmentof the samples. E. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition,will be subjected to an abbreviated gross necropsy examinationand all abnormalitieswill be recorded. Animals in a moribund conditionwill be anesthetizedwith sodium pentobarbital(via injectionin the marginal ear*'vein),bled via the vena cava, and exsanguinated,T.i'ssues,as describedin sectionE. Termination,i3) SampleCollection,will be collected. 0 00 J Page 23 of 25 TP8084.PK Page 8 (2) ScheduledSacrifice At approximately48 hours post-injection,animals survivingto terminationwill be anesthetizedwith sodium pentobarbita(lviainjectionin the marginalear vein), bled via the vena cava, and exsanguinated. An abbreviated gross necropsyexaminationwill not be done, however, tissueswill be collected. (3)SamRle Collection The whole liver and bile from each animaldying during the study,sacrificedin a moribundcondition,or survivingto terminationwill be collected. Both kidneysfrom each animalwill alsobe collected. The tissueswill be placed on dry ice immediatelyaftercollectionand then placed in a freezerset to maintaina temperatureof -20*C tlO*C. The tissues(liver,bile,kidneys)will be sent frozenon dry ice to the Sponsorafterexperimentaltermination. The sampleswill be shippedto the personlisted in Section7.D.(3).(c).The Sponsoris responsiblefor the retentionand dispositionof the samples. F. StatisticalAnalyses No statisticalanalysesare required. 8. Report A finalreportincludingthose itemslistedbelowwill be submitted. Descriptionof the test and controlmaterials Descriptionof the test system Procedures Dates of experimentalinitiationand termination Descriptionof any toxic effects Gross pathologyfindings(if applicable) Gross pathologyreport (if applicableand requestedby the Study Director) Page 24 of 25 TP8084.PK Page 9 9. Locationof Raw Data, Records,and Final Report Originaldata,or copiesthereof,will be availableat HWI to facilitateauditingthe studyduringits progressand before acceptanceof the finalreport. When the final reportis completed, all originalpaper data, includingthose item listedbelowwill be retainedin the archivesof HWI according to HWI SOP. Protocol and protocol amendments Dose preparationrecords In-liferecords Body weights Dose administration Observations Sample collectionrecords Shipping records Pathology Records Study correspondence Final report(originalsignedcopy) The followingsupportingrecordswill be retainedat HWI but will not be archivedwith the studydata. Animal receipt/acclimatiornecords Water analysisrecords Animal room temperature and humidity records Refrigeratorand freezertemperaturerecords Instrumentcalibrationand maintenancerecords Page 25 of 25 TP8084.PK Page 10 PROTOCOL APPROVAL 9-1- John L. Butenhoff, PhD Date Sponsor's Representative 3M Toxicology Service Medical Department Steven M. Glaza Date Study Director Acute Toxicology Hazleton Wisconsin,Inc. Representat,vgl Date Quality Assu nce Unit Hazleton Wisconsin,Inc. (6329-136.protdsk2) 0 0 0 lliOA-4 9.1.2 AnalyticalprotocolAMDT-010495.1 ()()()0"1:1 3M EnvironmentaLlaboratory Protoco-lAnalyticSatludy Single-DosIentravenouPsharmacokinetiSctudyofT-6053inRabbits In-VivoStudyReferenceNumber: HWI#6329-136 StudyNumber: AMDT-010495.1 TestSubstance:FC-99 (T-6053) Name and AddressofSponsor: 3M SCD Division 367 Grove Street St.Paul,MN 55106 Name and Address of Testing Facility: 3M EnvironmentalTechnology and Services 935 Bush Avenue St.Paul,NW 55106 Proposed InitiationDate: July25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers AMDT-M-1-0, AMDT-M-2-0, AMDT-M4-0, AMDT-M-5-0, AMDT-M-8-0, and Revisions: Thermal Extractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Extractionof Fluorochemicalsfrom RabbitLiver Analysisof RabbitLiverExtractforFluorochmicalsUsing ElectrosprayMass Spectrometry Analysisof FluorideUsing the SkalarSegmented Flow Analyzer with Ion SelectiveElectrode Author: James D. Johnson Approved By: meess D 6son St@mudy Diii-@ctor Date John Butenhoff,PhD Date Sponsor Representative 1.0PURPOSE To assestsheleveolfsystemiecxposurwehenT-6053isadministeraesadsingle intravenousinjectiotno rabbits. This studyisdesignedtoprovideinformationastowhetherthe perfluorooctanesulfonaatneiondoes go to therabbitliverand othertissueswhen the materialisadministeredinan intravenousdose and to ascertaitnhe change in concentratiownith time afterdose inserum and liver. 2.0 TIEST MATERIALS 2.1Test,Control,and Reference.Substancesand Matrices 2.1.1AnalyticalReference Substance: FC-95, lot161 or 171. They are equivalent. 2.1.2AnalyticalReference Matrix: Bovine liverand bovine serum 2.1.3AnalyticalControl Substance:None 2.1.4AnalyticalControl Matrix: Bovine liverand bovine serum 2.2 Source of Materials:3M ICP/PCP Division(2.1.1),grocerystore(2.1.22,.1.4 liver)S,igma Chemical Company (2.1.22,.1.4serum) 2.3Number of Testand Control Samples:Liverand serum from 4 testanimals and 1 controlanimal.Otherbiologicatlissue(skidneyb,ile,cellulafrractionw)ill be availableforanalysisifdeemed appropriatbey theStudy Director. 2.4IdentiflcatioonfTest and Control Samples: The samplesare identifieudsing theHWI animalidentificatinounmber which consistosf a lettearnd fivedigit number, plusthetissueidentitaynd day identit(yserum). 2.5Purity and Strengthof Reference Substance: To be determinedby Sponsor. 2.6StabilitoyfReference Substance:To be determinedby Sponsor. 2.7Storage ConditionsforTest Materials:Room temperature(2.1.1), -20 100C(2.1.2,.1.4)T.estandControslampleswillbe receiveadccordintgo ANMT-S-10-0. 2.8DispositionofSpecimens:Biologicatlissueasnd fluidswillbe retainepder GLP Regulationforthetimeperiodrequiredforstudieslongerthan28 days. This studyisinparallewlith a 28 day dermalabsorptionstudyso alltissueswillbe retained. 2 2.9SafetPyrecautionRse:fertoappropriaMtSeDS. Wearapproprialtaeboratory attireU.se cautionwhen handlingknivesforcuttingthe samples. 3.0 EXPERIMENTAL - Overview The tissuesfrom animalsdosed asdescribed(HWI#6329-136), areavailablefor analysisforfluorinecompounds. At thediscretioonftheStudy Directora, seriesof analyticatlestscanbe performed.The screeningforfluorideinliverviacombustion (See Methods--nextSection)istheappropriataenalysisto presentdefinitivdeatafor fluorinienthe liver.Sinceperfluorooctanesulfonaantieonisnot biotransformed, fluorinecontentwillbe an accuratestimateoftheconcentratioonfthe compound. 4.0 EXPEIUWNTAL -Methods 4.1Liver and Serum screeningmethods: (attached) 4.1.1AMDT-M-1-0, Thermal ExtractioonfFluorideby Means of a Modified DohrrnannDX2000 OrganicHalideAnalyzer-Liver 4.1.2AMMT-M-2-0, FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3AMDT-M-4-0, Extractionof Fluorochemicalsfrom RabbitLiver 4.1.4AMDT-M-5-0, AnalysisofRabbitLiverExtractforFluorochmicals Using ElectrosprayMass Spectrometry 4.1.5AMDT-M-8-0. Analysisof FluorideUsing theSkalarSegmented Analyzerwith Ion SelectivEelectrode 5.0DATA ANALYSIS 5.1Data Reporting: Data willbe reportedas a concentratio(nweight/weighto)f fluoridpeertissueorfluido,r asFC-95 (electrospramyass spectrometryp)erunitof tissueorfluid.Statistiucsed,atthediscretioonftheStudy Directorm,ay include regressionanalysisof serum concentrationwsithtimeand averagesand standard deviationosf concentrationfsordifferendtose groups.Ifnecessary,simple statistictaelstssuch as the Student'sttestmay be appliedtodeterminestatistical difference. 3 6.0MAINTENANCE OF RAW DATA AND RECORDS 6.1Raw DataandRecords:Raw dataa,pprovepdrotocoalp,propriastpeecimens, approvedfinalreporta,nd electronidcatawillbe maintainedintheAMDT archives. 7.0 REFERENCES 7.1AMDT-S-10-0, Sample TrackingSystem 8.0 ATTACHMENTS 8.1AMDT-M-1-0, Thermal ExtractionofFluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 8.2AMDT-M-2-0. FluorideMeasurement by Means ofan Orion EA940 ExpandableIon Analyzer 8.3AMDT-M4-0, Extractionof Fluorochemicalsfrom RabbitLiver 8.4AMDT-M-5-0. AnalysisofRabbitLiverExtractforFluorochmicalsUsing ElectrosprayMass Spectrometry 8.5ANMT-M-8-0, Analysisof FluorideUsing theSkalarSegmented Flow Analyzer withIon SelectivEelectrode 0' -ij4 3M Environmental Laboratory Method TherrnalExtractionof Fluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer - Liver Method IdentificatiNounmber: AMDT-M-1 RevisionNumber: 0 AdoptionDate: RevisionDate: None Author:RichYoungblom Approved by: /3 ?r4o'urlopu@pLL-eea.@,K/ Daie QualityAssurance Date Software:MS Word 5.1a AffectedDocuments: AMDT-M-2 FluoridMeeasurementby Means of an OrionEA940 Expandable[on Analyzer AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalideAnalyzer 1.0 SCOPE .APPLICABLE COMPOUNDS, AND MATRICES 1.1Scope:Thismethod isfortheoperatioonfa Dohnnann DX2000 when itisused toextract fluoridferom variousmatrices.The fluoridiestypicallcyollecteidnTISAB solutiofnoranalysis withan ionselectiveelectrode. 1.2ApplicableCompounds: Fluorochen-dcalosrotherfluorinatecdompounds. 1.3Matrices:Biologicatlissuesp,articularlliyver. 2.0 KEYWORDS 2.1Fluoridef,luorinex,tractiopny,rolysisi,onizatioino,nselectiveelectrodeD,ohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS 3.1Glasswareand exhaustgasescan be extremelyhot. 3.2Glasswareisfragileb,rokenglassmay causeinjuries. 3.3Pressurizegdases,propercompressedgashandlingpracticersequired. 3.4Solventbasedsamplesmay flashm,ay needtoallowthem todrydown beforestartinrgun. 3.5Potentiabliohazardsdue tothebiologicamlatrices.Use appropriatpeersonalprotective equipment. 4.0 SUPPLIES AND MATERIALS 4.1Compressed Oxygen, Hydrocarbonfree,regulatetdo30 PSI. 4.2Compressed Helium,High PurityGrade,regulatetdo45 PSI. 4.3QuartzglasssampleboatwithTeflonTMtubingD,ohrmann 890-097orequivalent. 4.4Quartzglasscombustiontube,RelianceGlass G-9405-012 orequivalent. 4.5Onon 940999 TotalIonicStrengtAhdjustmentBuffer(TISAB II)or equivalent. 4.6Sample collectiovnialsH,DPE. 4.7Milli-QTMwater 4.8Polystyrenepipettes. 4.9ActivatedCharcoal,E.Merck 2005 orequivalent. 4.10HamiltonSyringeor equivalent. 4.11Miscellaneouslaboratorgylassware 5.0 EQUIPMENT 5.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzer,modifiedforfluorideextraction. 5.2IBM compatible386 or486 computer. 5.3DX2000 softwarev,ersion1.00,modifiedforfluor-iedxetraction. 5.4Excel Spreadsheetv,ersion5.0orgreater 6.0 INTERFERENCES 6.1 Sample sizeislimitetdoapproximately150mg, dependingon samplemoisturecontent.This may varyfrom matrixtomatrix. 2 ()()003!1 7.0 SAMPLE HANDLING 7.1Samples arenottobe handledwithbarehands.Fluoridemay leachfrom theskintothe sample. Use forcepsor probetotransfetrissues. 7.2Samples ofliverarecutfrom frozenliverand placedina taredand labeledweigh boat.Use a cleanscalpeland cuttinbgoard.The cuttinbgoardand scalpeslhouldbe cleanedwithwater, methanol,or methanol-watesrolutioanftereach liveriscut. 8.0 CALIBRATION AND - TANDARDIZATION 8.1PreparationfCaUbratioSntandards 8.1.T1hestandarrdesquirfeodreachprojecwtilnleedtobeapproprifaotrethaitndividuparloject. Refertoprotocolforthatproject. 8.1.2Typically50-500 ppm FC-95 inmethanolstandardsareused. 8.1.3Forrabbitlivesrtudiesu,sebeefliverasthematrix.Cut a pieceoffrozenbeeflive(r100150mg) and weigh itina labeledandtaredweigh boat. 8.2 Calibration-Overview The normal calibratiiosnthefluoridceurve(AMDT-M-2). However, ifan optionalspikedliver curveisrequiredtheprocedurelistebdelow isused. 8.2.1A calibraticounrvefortheDX2000 isgeneratebdy spaffigsampleswithknown standards and combustingthem usingthesame methods and matrixtypeasthesamplestobe tested. 8.2.2Typicallyt,hreereplicateosfeachstandardand fiveconcentratioonfsstandardswillbe spiked. 8.2.3Standardcurvewillbe plotteadsMass SpikedF (ug)on thex-axisand StandardMass RecoveredF (ug)on they-axis.Generatea regressiocnurveand calculatteheequationfortheline and ther2 value. 8.2.4Mass SpikedF (ug)= (Amount spikedinn-LLx) (Conc.ofstandardinppm) x (0.6004)* *FC-95 is60.04% F therefor0e.6004isthefactoursedtoconvertFC-95 toF 8.2.5StandardMass RecoveredF (ug)= (TISAB volumeinmL) x (Orionreadinginppm) 8.3Calibratio-nProcedure 8.3.1StartUp 8.3.1.1Run 2 ormore CleanCycleswhen startinignstrumenetachday.More cleancyclesmay be usediftheprevioussamplescontainedhighconcentratioonfs fluoride. 8.3.2Blanks 8.3.2.1Preparesampleusingthesame methods and typeofmatrixasthetestsample. 8.3.2.2Forrabbitstudiesu,sebeefliverasthematrix.Prepareatleast3 samplesofbeefliver (100- 150 mg) forblanks. 8.3.2.3Putsample inDohrrnannboat.Combust eachsampleasdescribedinsection9.0and analyzesampleaccordingtomethod AMDT-M-2 fortheionselectiveelectrodaenalysis. 3 0()00&1+() 8.3.2.4For rabbitstudiest,he meter readingfora blank sample should be 0.03 ppm or lower beforeproceedingwith thecalibrationB.um samples untilthislimitisreached,or untilinthe judgement of theoperatorthereadingisstablewith respecttohistoricarleadings(previous48 hours). 8.3.2.5For non-rabbisttudiest,heblankreadingsshouldreacha predeterminedionconcentration beforeproceedingwiththe calibration. 8.3.2.6Itmay be necessarytomix approximately50 mg of charcoalwith thesample to aid combustion. 8.3.3 Standard Curve 8.3.3.1Weigh out atleast15 matrixsamples (5 standardswith3 replicateesach)in taredand labeledweigh boats.For rabbitstudiesw,eigh 100-150 mg beefliversamples. Record weightsin studydata. Storethematrix samples on dry iceor icepacks tokeep them frozenuntilused. 8.3.3.2 Place weighed beef liversample inDohrmann sample boat. 8.3.3.3Startwith theloweststandardconcentration.Using a Hamilton syringe,ejecta fixed quantityof the standardon orinthematrix. For rabbitstudies,use 4 uL of standardand ejectiton or inthebeefliver. 8.3.3.4At least3 replicatesshouldbe used forthelowest standardconcentrationm;ore replicates may be used atthediscretioonf theanalyst. 8.3.3.5Combust the sample as describedin section9.3 and analyzeaccordingtoAMDT-M-2. 8.3.3.6Run all15 standards.Ifone replicatiessignificantldyifferenftrom theothertwo replicatesn,m anothersample forthatstandard.Indicateindatathatthenew replicatreeplacesthe old replicateand thatthenew replicatweillbe used to calculatetheregressioncurve. 8.3.3.7When allstandardshave been ran,calculattehe r2. r2 must be atleast0.95.Ifitisnot at least0.95,conswt with supervisor. 8.3.3.8A new standardcurve shouldbe run when thecombustion tubeor sample matrixis changed. New standardcurvemay alsobe run atthediscretionof theanalyst. 8.4 Storage Conditions for Standards 8.4.1Storage requirementsforstandardsaredependent on the individualstandardsused. Typically,standardsarestoredatroom temperatureinplasticscrew top bottles. 8.4.2 New FC-95 standardsshouldbe preparedatleastonce a month. 9.0 PROCEDURES 9.1TypicalOperating Conditions: 9.1.1Combustion tubetemperature= 950'C. 9.1.2Oxygen and Helium flow = 50 cc/minute. 9.1.3Vapoiization/Dryingtime = 240 seconds. 9.1.4Bake time = 300 seconds. 9.2StartUp Procedure: 9.2.1Ifthe program isnot starteds,tarthe EOX program on thePC. 9.2.2Open the SYSTEM SETUP window. 9.2.3Put the furnacemodule and thecellinthe READY mode. 9.2.4Close the SYSTEM SETUP window. 4 9.2.5When the oven has reachedtheREADY temperature,run the CLEAN found in theCELL CHECK menu. 9.2.6 See AMDT-EP-3 fordetailsof theDohrmann software. BOAT program 9.3Sample ExtractionProcedure: 9.3.1Open theSAMPLE HATCH and placethesample intheBOAT. It may be necessary to nlixapproximately50 mg of charcoalwith the sample toaid combustion.' Ifthisisdone, charcoal should alsobe mixed inwhile establishintghebaselineand when generatingthestandardcurve. 9.3.2Close SAMPLE HATCH. 9.3.3Add appropriatevolume of TISAB solutionor 1:1 TISAB:Milli-QTIlwater mixture toa labeledsample collectiovnial.Typically0.6mL to 15 niL areused. For rabbitstudiesu,se 1.0or 2.0mL of 1:1 TISAB:Milli-QTNIwatermixture. 9.3.4 Placethevialso thatthetipof theCOMBUSTION TUBE isintheTISAB atleast0.25 inches.Gases releasedduringpyrolysismust bubble through theTISAB. 9.3.5 Run theEOX-SOLIDS program found in theRUN menu. 9.3.6When theEOX program isfinishedr,emove the collectiovnialfrom the combustion tube. 9.3.7IfundilutedTISAB was used tocollectthesample,add an equalvolume of Milli-QTm water to theTISAB tomake 1:1 TISAB:Milli-QTM. 9.3.8Rinse the end of the combustion tube with Milli-QTMwaterand wipe with a KIMWIPE to remove any TISAB remainingon thetube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cottontippedapplicatororvacuumed out. Itmay be necessarytoscrapparticleosffthe bottom with a spatulaorothersimilardevice.A drop of Milli-QTMwater may be added tothe boatto aidinthe Clean Cycle. 9.3.10 Close thehatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12Sample isreadyforanalysisby ionselectiveelectrode(ANMT-M-2). 9.4 Sample Calculations 9.4.1 Use the standardcurveto calculatethesample value. 9.4.2Sample Mass Recovered F (ug)= (TISAB volin mL) x (Orionre dinizin12pm - interc=t) (Slope) 10.0 VALIDATION 10.1QualityControl 10.1.D1ailyStartUp Check Samples:Once thestandarcdurveisestablisheadc,hdayof analysisisstartedby analyzingQC samples. The QC samples aretobe the same asthe lowest concentrationspiked samples used togeneratethestandardcurve. Each concentrationmust be done intriplicatuenlessthe firsttwo replicateasrewithin20% ofthe standardcurve,then a third replicatiesnot necessary. 10.2 Precisionand Accuracy: See method development analysisand sample analysisin FluorideNotebooks 2,3,and 5. Precisionand accuracyvarieswhen analyzingsamples of different matricesand differentreferencecompounds. 10.3 Other ValidationParameters: NA 5 0000,10*t.>r 11.0 DATA ANALYSIS- 11.1 Calculations 11.1.1For thestandardcurve,useregressioannalysiisnExcel,version5.0or greater. 11.1.2To calculattehefluoridceontractioinnthesample,seemethod AMDT-M-2. 11.2 Analyzing theData 11.2.1r2must be atleast0.95orgreater".Outliersm"ay be excludediftwo ofthethreereplicates arewithin20% ofeachotherand theoutlieirsgreatetrhan200% oftheaverageofthosetwo or lessthan50% oftheaverageofthosetwo. Any suchoutliersshouldbe pointedoutinthedataand notedintheFinalReportalongwiththereasonitwas consideredan outlier. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1Rosemount Dohrmann DX2000 OrganicHalideAnalyzerOperator'Msanual (Manual915349,revisioBn, December 1993) 13.2 ANTDT-M-2 FluorideMeasurement by Means ofan OrionEA940 ExpandableIon Analyzer 13.3AMDT-EP-3 RoutineMaintenanceofa ModifiedDohrmann DX2000 OrganicHalide Analyzer 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 6 0()00,,I-l 3M E aboratory Method FluorideMeasurement by Means of an Orion EA940 Expandable Ion Analyzer Method IdentificatioNnumber: AMDT-M-2 RevisionNumber: 0 AdoptionDate: /0-4-fsRevisionDate: None Author. Rich Youngblom Approved By: G@, Leader Date 3@IL-Z QualityAs-surance /,o- 4c--@Date Software: MS Word 5.1a AffectedDocuments: ANMT-M-1 Thermal Extractioonf Fluorideby Means of a Modified Dohrrnann DX2000 Organic HalideAnalyzer 0 OIAT 1.0 SCOPE, APPLICABLE COMPOUNDS, AND MATRICES I.ISCOPE: Thismethod isforthecalibratiaonnd operatioonfan OrionEA940 Expandable IonAnalyzer. 1.2APPLICABLE COMPOUNDS: Fluoride. 1.3APPLICABLE MATRICES: Liquidsamplesinan appropriatbeuffersolutionP.referred pH of 6.0. 2.0 KEYWORDS 2.1Fluoridef,luorinei,on selectiveelectrode 3.0 PRECAUTIONS 3.1No hazardisdentifwiietdhthimsethod. 4.0 SUPPLIES AND MATERIALS 4.1Orion940999 TotalIonicStrengtAhdjustmentBufferH (TISABII)orequivalent. 4.2OrionModel 900001 electrodfeillinsgolutio(nAgCl)orequivalent. 4.3Orion940907 100 ppm fluoridsetandardorequivalent. 4.4Milli-QTIwIaIteror equivalent. 4.5Magneticstirbars. 4.6Lab tissues. 4.7Sample collectiovnials. 4.8Plasti1c00 mL volumetricflasks. 4.9Polystyrenepipettes. 4.10Miscellaneouslaboratorgylassware. 5.0 EQUIPMENT 5.1OrionModel EA940 ExpandableIonAnalyzerorequivalent. 5.2OrionModel 960900 SolidStateCombinationFluoridelectrodoerequivalent. 5.3MagneticStirPlate. 5.4IBM compatible386 or486 computer(onlyneededifusingOrion3E software). 5.5OrionRS232 interfacceable(onlyneededifusingOrion3E software). 5.6MicrosoftExcel5.0(onlyneededifusingOrion3E software). 6.0 INTERFERENCES 6.1Itisrecommended thatthepH be atornear6.0.A I-IrnixturoefTISAB and samplenilliQTM waterwillgenerallybringsample topH of6.0. 6.2Sample temperaturmeay effectfluoridmeeasurement. Itisrecommended thatthesample be atroom temperatureas thestandardswere when themeterwas calibrated. 6.3The ratethesamplesarestirreadtshouldbe consistenwtiththeratethestandardswere stirred. 2 oooo,-Ir%I- 6.4Air bubblestrappedunder electrodcean giveerroneousreadings.Make sureno airistrapped under electrode. 7.0 SAMPLE HANDLING 7.1 No specialhandlingnecessary. 8.0 CALIBRATION AND STANDARDIZATION 8.1PreparatioonfCaUbratioSntandards 8.1.M1easure50mL ofTISAB IIinto5 100mL plastivcolumetrfilcasks. 8.1.2Labeltheflaskass0.050,.10,.5,1.0a,nd1.5ppm F-,alongwiththedateandyourinitials. 8.1.P3ipett0e.050,.1,0.5,1.0a,nd1.5mL of100ppm fluorisdteandaridntotheappropriately labeledflasks. 8.1.4Add approximately30 niL of Milli-QTMwaterto each flask. 8.1.5Shake theflaskstoniixthesolutions. 8.1.6Eliminateairbubblesfrom theflasksby tippingtheflaskosn theirsidesand rollingtheairin theflasksover theairbubbles. 8.1.7Bringthevolume in theflasksup tothe 100 mL mark withMilli-QTMwater. 8.1.8Invertand shakethe flasksforthefinalmixing. 8.1.9Record standardsinStandardsLog Book. 8.2 Calibration 8.2.1Ifnecessary,remove tapefrom electrodfeillinhgole. 8.2.2Invertprobe towet top seal. 8.2.3Ejecta few dropsof fiflinsgolutionfrom bottom of electrodteo wet lowerseal. 8.2.4Fidltheelectrodweith fillinsgolution. 8.2.5The meter and theF- electrodaeretypicallcyalibratebdy directmeasurement withno blank correctionu,singstandardswith concentrationosf0.05,0.1,0.5,1.0,and 1.5ppm F-,following themanufacturer'sinstructions. 8.2.6Record theslopein theappropriatelogbook. 8.2.7Cleantheelectrodbey rinsingwithMilli-QTMwaterand wipingthesidesdown withlab tissues. 8.3 Storage Conditions for Standards 8.3.1Calibrationstandardsare storedatroom temperature. 9.0 PROCEDURES 9.1Calibrationand Measurement, Standard method: 9.1.1The sample tobe measured needs tobe n-dxedwithTISAB usingtheproportions recommended by theTISAB manufacturer. 9.1.2Placea st@barinthesampleand placethesampleon thestiprlate. 9.1.3Allow thesample to n-iifxora few secondsbeforeinsertintgheelectrode.When the electrodeisinsertedm,ake surethereareno airbubblestrappedunder theelectrode. 9.1.4The sample shouldbe thesame temperatureas thecalibradonstandardsand stirreadtthe same rateas thecalibratiosntandards. 9.1.5When thereadingshave stabilizerde,cordthereadingintheappropriatleogbook. 3 ()()004()' 9.2CalibrationAnd Measurement, Using Orion 3E Software: 9.2.1Calibration: 9.2.1.1Follow steps8.2.1to8.2.4. 9.2.1.2PressFunctionKey #8 (F8). 9.2.1.3The computer screenwillask you tocon firmthenumber of standardsto be used, concentrationof thestandards,and whether ornot a blank istobe includedinthe calibration. Make any necessarychanges totheinfonnationpresentedand clickon CONTINUE. 9.2.1.4Placethe electrodein thefirssttandardon thestirplateand clickon CONTINUE. 9.2.1.5Observe thereadingson the graphicdisplayon thecomputer. When the readingshave stabilizedp,ressACCEPT READING. 9.2.1.6Repeat step9.2.1.4and 9.2.1.5forthe remaining standards. 9.2.1.7Afterthe finalstandard,thecomputer willdisplaythe slopeof thecurve,as well asthe intercepatnd correlationR.ecord theslope,intercepta,nd correlatioinnthe appropriatelog book and clickon CONTINUE. The calibratiodnataisautomaticallcyopiedto C:\Orion\Data\Calib.txt. 9.2.2Data Spreadsheet: 9.2.2.1SelecteitherNEW or OPEN from theFILE menu toopen a new or existingspreadsheet to storedata in. 9.2.2.2Record thename of the spreadsheetused intheappropriatelog book. 9.2.3Fluoride Measurement: 9.2.3.1Follow steps9.2.1through 9.2.4 9.2.3.2Enter thename of thesample in theappropriateplaceon thescreen. 9.2.3.3Click on the NEW SAND[PLE button 9.2.3.4When thereadingshave stabilizedc,lickon the RECORD buttonand writetheresudtinthe appropriatelog book. 10.0 VALIDATION 10.1 QualityControl: 10.2 Precisionand Accuracy 10.3 Other ValidationParameters According toReference 13.2,therangeof detectionis0.02 ppm fluorideup toa saturatedsolutionoffluoride. 11.0 DATA ANALYSIS 11.1 CalculationsNone necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS None 13.0 REFERENCES 4 13.1Orion Model EA940 Expandable IonAnalyzerInstructioMnanual,Orion Research Incorporated1,991. 13.2Orion Model 960900 SolidStateCombinationFluorideElectrodeInstructioMnanual,Orion Research Incorporated1,991. 14.0 REVISIONS Revision Numbe Reason forChange Revision Date 5 ()0004,S 3M Environmental Laboratory Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP IdentiricatioNnumber: AMDT-M-5 Revision Number: 0 Adoption Date: RevisionDate: None Author: Dave Christenson/CynthiWaeber Approved By: @:p roup LLeeaadderer Date QualityAssurance Date Software:MS Word, 6.0 AffectedDocuments: M-4, ExtractionofFluorochernicalfsrom RabbitLivers 0()001-1!) 1.0 SCOPE 1.1 Scope: Thismethod isfortheanalysisofextractsofrabbitliveror othertissueosr fluidsforfluorochemicalsusingthe electrospramyass spectrometerT.he analysis isperformed by singleion monitoringofFC-95 anion,M/Z= 499, theinternal standardM/Z = 427, and otherappropriatemasses. 1.2 Applicable Compounds: Fluorochemicalsor otherfluorinatedcompounds. 1.3 Matrices: RabbitLivers(samples),Beef Liver(standards)o,thertissuesand fluids. 2.0 KEYWORDS 2.1 Fluorochemicals,fluorinatecdompounds, electrospramyass spectroscopy,mass spectrometer,rabbitlivers. 3.0 PRECAUTIONS 3.1 Use cautionwith thevoltagecablefortheprobe.When the voltagecableisplugged intotheprobe DO NOT TOUCH THE PROBE, thereisriskof electricaslhock. 3.2 Do not run thepump above it'scapacityof4000 psi.Ifpressuregoes over 4000 psi stopand releasepressure.The peak tubingmay be plugged.Troubleshoot back to findtheplug and replacetheplugged tubing.See AMDT-EP-15 3.3 Do not run the pump todryness. 4.0 SUPPLIES-AND MATERIALS 4.1 Supplies 4.1.1 Nitrogengasregulatetdo 140 psi. 4.1.2 Fluofixcolumn or equivalent. 4.1.3 100 uL or250 uL flattipsyringeforsample injection. 4.2 Reagents 4. 2.1 Diluteacetonitrimloebilephase,diluteacetonitri1l:e1 with Milli-Qwater. 4.2. 2 Milli-Qwater,allwaterused inthismethod shouldbe Milli-Qwater. 5.0 EQUIPMENT 5.1 VG Trio2000 ElectrospraMyass Spectrometeorrequivalent. 5.2 ISCO SyringePump 5.3 SpectraphysicsAS300 Autosampler 5.4 100 uL Assembly 5.5 Autovialsor capped centrifugetubes. 6.0 INTERFERENCES 6.1 There areno known interferenceastthistime. 7.0 SAMPLE HANDLING 7.1 Keep theextractedsamples incapped 15 mL centrifugetubesor incapped autovials untilready foranalysis. 0()005() 2 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8. 1.1 Seven beefliverstandardsand one blank beefliverarepreparedduring the extractiopnrocedure.(See AMDT-M-4, section8.0) 8.2 Calibration 8. 2. 1 Run the seven beefliverstandardstwice,startinwgith the loweststandard to obtainthe standardcurve. 8. 2.2 Typicallyone standardisrun aftereach 5 to7 samples.Choose a standard in the same range of concentrationas thesamples. 8.3 Storage Conditions for Standards 8.3. 1 Fresh standardsareprepared with each analysis.Standards arestoredin covered plasticentrifugetubesuntiltheanalysison themass spectometeris performed. Samples and standardsare NOT reftigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8.4. 1 Beef liverhomogenates may be frozen afterpreparation. 9.0 PROCEDURE 9.1 InitialSet-up 9.1.1 Set softwareto"Operateon", Ion Mode ES-. 9.1.2 Record backing pressureintheinstrumentlog. 9.1.3 Fillthesolventcylinderwith mobile phase. 9.1.4 Set the pump to"Run". Set theflow to 1000 uL/min. Observes droplets coming out of thetipof theprobe.The pressureshould be at 1700 to 1800 psi. 9.1.5 Check thefused silicattheend of theprobe.Use an eye piecetocheck for chips.The tipshouldbe flatwith no jagged edges.Ifany chipsare found cutoffthetipofthesilicawitha column cutterand pullthe silictahrough to theappropriatelength. 9.1.6 Check your nitrogensupply.Turn on thenitrogen.There should be no nitrogenleakingaround the tipof theprobe.A finemist shouldbe coming out of thetip. 9.1.7 Carefullyguidetheprobe intotheopening.Insertituntilitwon't go any furtherC.onnect thevoltagecabletotheprobe. 9. 1.8 Go tothe"Editor"page,and setIonizatioMnode toES-,and the appropriatemasses to 427 and 499. 9.1.9 Ifitisnot insingleion mode go to"Option" and setSIR. 9.1. 10 StartAcquisitionA.ssign a filename, MO-DAY-YR + letterR.ecord itin the log book. 9. 1. 11 Run thebeefliversamples firstr,unning each standardtwice atthe beginningof therun..Run a QC check by running one standardafterevery 5 to7 samples. 9.2 Manual Injection 9.2. 1 Draw 150 uL of sample intoa syringe.Injectthesample intotherheodyne injectiopnort.Injectslowly.Record thesample ID inthe log book. 9.2.2 Turn thevalveto"On". 9.2.3 Wait two minutes,and injecthenext sample. 9.2.4 Record thescan number foreach sample inthelogbook. 0()()051 3 9.3 Using the Autosampler 9.3.1SetupsampletrayA,B,orC. 9.3.2 Record thesamplesand theirpositionisn theinstrumentlog book.Up to 17 vialsmay be ineach run. 9.3.3 Set-upthesampler: 9.3.3.1 Push thesample button 9.3.3.2 Setsample loop size= 100 uL 9.3.3.3 Setinject/sampl=e2 9.3.3.4 SetCycle time = 0 9.3.3.5 Name thefileL:ivers 9.3.3.6 Identifythetrayused 9.3.3.7 Add thesamplestoQueue by pressing"Enter" 9.3.3.8 Press"Run" tostart 10.0 VALIDATION 10.1 QualityControl 10.1.1Run a standaredvery5 to7 samplesI.fa significacnhtange(+5-0%) in peak heightoccursstoptherun.Only thesamplesbeforethelastacceptable standardwillbe used.The remainingsampleswillbe reanalyzed. 10.2 Precision and Accuracy 10. 2.1 See Method ValidationReportnumber AMDT-M-5.0. V 1 10.3 Other Validation Parameters 10.4 Referto Method ValidationReportNumber AMDT-M-5.0.Vl 11.0 DATA ANALYSIS 11.1 11.2 Calculations Plotthestandardcurve,usingthemean of thetwo valuesobtainedforeach standard. 11.2. 1 Read peak heightsor areasforthesamplesfrom theprintoutU.se linear regressiontodeterminethesample concentrations. 11.2.2 Calculatethemg of FC-95 anion,or otherfluorochemicalinthetotalrabbit liver: mg FC-95 anioninthetotarlabbitliver mizFC-95 anionfrom std.curve gms of liverused foranalysis x Totalmass of liverg,ms 11.3 Make a resulttsableand enteritinthestudybook. 11.4 Printachromatogram foreach sample,withthepeakslabeledwith thesampleor standardDD.Write thestudynumber on theprintouti,nitiadla,te,and put itinthe studyfolder.Stapleallchromatobrramstogetherand number pages. OJ 4 I 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-EP- 17 14.0 REVISIONS Revision Number Reason forchanze I -. . -- - Revision Date 0 ()0 0 ro",I5 3M Environmental Laboratory Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion SelectiveElectrode Method IdentiricatioNnumber: AMDT-M-8 Adoption Date: Revision Number: 0 Revision Date: None Author: Deb Wright/ CynthiaWeber Approved By: ?up up L2e2aed@ear@d Date QualityAssurance Date Software:IBM MS Word, 6.0 AffectedDocuments: AMDT-EP-26, Analyzer Operationand Maintenance of theSkalarSegmented Flow 0()00511 1 1.0 SCOPE 1.1 This method isfortheanalysisforfluoridet,hermallyextractedfrom samples using theDohrmann DX2000 (AMDT-M-1), and collecteidnTISAB foranalysiwsithan IonSelectivEelectrode(ISE).The analysisisperformedusingtheSkalar Segmented Flow AnalyzerwithISE. 1.2 Samples can be tissuess,erum,biologicamlaterialo,r othermaterialsextractedon the Dohrmann. 2.0 KEYWORDS 2.1 Skalar,segmented flow,fluoride. 3.0 PRE. CAUTIONS - 3 I Follow standardlaboratorsyafetypractices. 4.0 SUPPLIES AND -MATERIALS 4.1 Supplies 4.1.1 Sample i-.ups,4 mL plasticcupswithcaps 4.1.2 Autopipetso,xfordor equivalenwtithplastitcips 4.1.3 Polypropylenevolumetricflasks1,00 mL 4.1.4 Cartridgecomponents,refertotheSkalarMethods forcomponents and part numbers. 4.1.5 Sample prefilterEsv,ergreen 4.2 Reagents 4.2.1 Brij35,30% S.F.A.S.Detergent 4.2.2 TISAB IIbuffersolutionP:urchaseTISAB IIfrom Orion.To I liteorf TISAB IIadd 2.5mL or 100 ppm fluoridseolutionand 1 n-LLBrij. 4.2.3 SamplerrinsingsolutionD:iluteTISAB 111:1 withMilli-Qwater. 4.2.4 Nitricacidsolutiofnordecontamination1,N (labgade): Slowlyadd 64 mLs concentratenditriaccid(HN03)to 250 mLs of Milli-Qwater.Bring thevolume up toIL withMilli-Qwater. 4.3 Standards 4.3.1 Stock solution1,00 ppm F: purchasedfrom Orion. 4.3.2 Intermediatsetandard1,0 ppm: Dilute10 mLs of stocksolutionto 100 mLs withMilli-Qwater.Use polypropylenevolumetricflasks. 4.3.3 Worldng standardM:ake up thefollowingworking standardsby addingthe volumes ofintermediatoer stockstandardindicateodn thetable,using oxfordorpumpmate pipets,to50 mLs of TISAB and dilutingto 100 mLs withMilli-Qwater. Working Standard mLs ofStock Standard 0.015 ppm 0.03 ppm 0.06 ppm 0.09 ppm 0.12 ppm 0.15 ppm 0.3 ppm 0.3 mLs of IntermediatSetandarei 0.15 0.3 0.6 0.9 1.2 1.5 - 0.6 ppm 0.6 1 2 0 0 "r@5 1.2ppm 1.2 1.5ppm 1.5 5.0 EQUIPMENT 5.1 Skalar Segmented Flow Auto Analyzer Sans"' System equipped with ISE 6.0 INTERFERENCES 6.1 High concentrationosf alkalinityc,hloridep,hosphate,sulfateor ironcan cause interferences. 7.0 SAMPLE HANDLING 7.1 Samples should be storedinpolyethylenebottlesS.amples should be analyzed within30 days. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of CalibrationStandards 8. 1.1 Prepare calibratiosntandardsas in section4.3. 8.2 Calibration 8.2. 1 The standardsare analyzedatthebeginningof therun. 8.3 Storage Conditions for Standards 8.3. 1 Standardsarestoredincapped polypropylenevolumeuic flasks.New standardsareprepared ata minimum ofevery sixmonths, or as necessary. 9.0 PROCEDURE 9.1 Start Up Procedure 9.1.1 Clamp down thepumpdecks, airbarsand sampler-pump tubing. 9.1.2 Put the fluoridelectrodesintheelectrodechamber. 9.1.3 Turn on thepower of the sampler,pumps, offsetpotentiometerand heating bath. 9.1.4 Put therea-ent-lineisntheappropriatebottles. 9.1.5 Turn on theinterfacec,omputer,displayand printerM.ake turn on the interfacebefore the computer. sure you 9.1.6 Letthesystemstabilifzoerapproximatel3y0 minutes. 9.2 Startinga Run 9.2.1 Createa sample tableby selectinFgILES, TABLE, and CREATE, typein the name of the filea,nd pressENTER. 9.2.2 Printthesampletablei,nserteidnthesystemtableby pushingESC, PRINT, GROUP 1. This will print the entire run. 9.2.3 Dial thesampler settingstotheappropriatenumber of samples,number of seconds forsample wash, and number of seconds for the sample. 9.2.4 Fillthesample traywith thestandirds,samples,washes and driftsr.W and FW/RUNOUT cups on the samplerdo not need tobe filled. 9.2.5 Set thebaseline. 3 000o.ri-6 9.2.5. 1SelectGRAPHICS, REAL TIME. Ifyou cannotgetreal-timey,ou may be intheData Handling Panel.Switch tothe AnalysisPanel by selectingCONTROL PANEL and pushincrF7. 9. 2.5.2 Use thesmallscrewdriverfortheoffsetpotentiometertosetthe base line.Adjustthebaselineuntilitisapproximately3/4 inch from the bottom of thescreen. 9.2.5.3Check thehigheststandardand adjustthe gain,ifnecessary,with theinterfacescrew #3. 9.2.6 Go toCONTROL PANEL, and toanalysispanel.Deselecttheanalysisthat willnot be run.(Selector deselectanalysisby pressingENTER.) PressTab to returnto theAnalysisPanel. 9.2.7 Pressthe spacebartobrin-up thelocalmenu. 9.2.8 SelectSTART tostarthe analysis. 9.2.9 Type your ID (initialst)h,esample tablewhich you createdunder 9.2.1(or pressENTER forchoices),choose running with or withoutthe system table and selectSTART ANALYSIS. 9.2. 1OAfter startingthesoftware,starthe sampler.Make surethatthesampler is settotherightnumber of samples and thatthe sample/wash/airtimesare OK. 9.2. 11 SelectGRAPHICS, REAL TIME toview theprogressof theanalysis. 9.3 Loading and Printingthe Data-File 9.3.1 Go toCONTROL PANEL, pressthe spacebartobring up thelocalmenu and selectLOAD. SelectAUTOCALCULATION and enterthefilename(or highlightthe filetobe printedand pressENTER). 9.3.2 To view thecalibratiocnurve,go toGRAPHICS, CALIBRATION CURVE. 9.3.3 To printthe high levelcurve,push PRINT SCREEN. 9.3.4 To printthelow levelscreen,push !ESC togetout of graphics.Select SETTINGS. Change themax y valuetoapproximately900. Go toCAL CURVE and pressESC, and Enter.PressPRINT SCREEN. 9.3.5 Return toSETTINGS and change themax value back to4095, go toEDIT, pressENTER and PRINT SCREEN toprintsample peaks. , 9.3.6 To printtheresultgso toCONTROL PANEL, SPACEBAR OUTPUT, OUTPUT. SelectPRINTER fortheEpson or PRN for theLaser. 9.4 Shutdown 9.4.1 Put allthere:agent-liniensMilli-Qwater. 9.4.2 Let the system rinseforapproximately30 minutes. 9.4.3 After thesystem has rinsedcompletely,turnoffthe sampler,pump and offsetpotentiometerT.urn offtheheatingbathon weekends. Leave liquidin the lines. 9.4.4 Take the electrodeout and soak in 100 ppm F overnight. 9.4.5 Release thepump-decks, airbirsand sampler pLtMp-tubin,-,. 9.4.6 SelectFILES, pressALT F and selectQUIT toexitthe pro,-,ram. 9.4.7 On Friday,turnoffthecomputer,displayand interfaceforthe weekend. 10.0 VALIDATION 10.1 Quality Control 10.1.1Run a standar(dmidtohighconcentratioenv)ery10samples.Ifa significancthange inpeak heiIahtoccurs,only thesamples before thelast acceptablestandardwillbe used.The remaining samples willbe reanalyzed. 4 0()0057 10.2 Precisionand Accuracy 10. 2. 1See Method ValidationReport number AMDT-M-8.0. V 1 10.3 Other Validation Parameters 10.4 Referto Method ValidationReport Number AMDT-M-8.0. V I 11.0 DATA 11.1 11.2 11.3 11.4 ANALYSIS Calculations 11. 1.IThe standardcurve isplottedby the Skalarsoftware. 11. 1.2AII calculationasredone by the Skalarsoftware.r2 should be 0.995or better. Prepare spreadsheetsto summarize data.Includesample volume, weights used etc. Write thestudy number on theprintoutsi,nitiald,atetheprintout,and bind together with allpackage documents and placeinthe studyfolder.Make a copy of the summary sheetand tapeintothestudy notebook.Back up alldataand spreadsheets onto study diskand backup disks. ElectroniDcata 11. 4. 1GLP studiesE:lectronicdataiscopiedonto theStudy floppydiskforeach study,and alsodataiscopied onto a floppydisk thatisstoredinthelab. 11. 4.2 Other studiesA:ll dataiscopied onto a floppydiskthatisstoredinthelab. 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 ANDDT-M- 1,Thermal Extracdon ofFluorideby Means of a Modified Dohrmann DX2000 OrganicHalideAnalyzer-Liver 13.2 SkalarMethods, #335, SkalarMethods Manual 13.3 ANIDT-EP-26, Operationand Maintenance of theSkalarSegmented Flow Analyzer 14.0 REVISIONS Revision Numbe Reason forchang!; Revision Date 5 @- I . @i @ 1 ' 9.3 QualityAssurance Unit Statement 0()()05@) AttachmentD GLP Study QualityAssuranceStatement .......... Study Title: Study Number: Single-dose AMDT-010495.1 Intravenous Pharmacokinetic Study of T-6053 in Rabbits Name ofAuditor:KariRambo This studyhas been inspectedby theQualityAssuranceUnit asindicatedin thefollowingtable. The findingswere reportedtothestudydirectorand management. InspectionDates EM 12 10/13/95 10/19/95 Phase FinalReport Date InspectioRneportedto Manag=t StudyDirector 10/19/95 10/19/95 1 -It QAU Auditor Date @ -@ -- 1 .1.@@ - @. -47 @ @t 9.4 Key PersonnelInvolvedinthe Study 0()0061 3M Environmental Laboratory Key Personnel Thermal extractiofnollowedby analysiussingOrion Ionanalyzer: Jim Johnson Deb Wright RichYoungblom Deann Plummer Analysisofliverextractussingelectrospromyass spectrometry*, Jim Johnson Dave Christenson Thermal extractiofnollowedby analysiussingSkalarsegmentedflow analyzerwithIonselectiveelectrode: Jim Johnson Deb Wright RichYoungblom Deann Plummer Documentationand Reporting: Jim Johnson RichYoungblom QualityAssuranceUnit: GaleVan Buskirk CynthiaWeber KariRambo 00006;,@b I -I., @- @- .@@ . @-.@-., . I - I - @t@@, -:@;f-@ 9.11Data ()()OOG;l ,-, .. qzwm@ @ 9.11.1Summary and raw data;ug F -inwhole liveras determinedby thermalextraction followedby analysisusingOrion ion analyzer. 0()()O(')I Summary ofCombustion Data -Liver AMDT-010495.1, HWI 6329-136 As Referenced in FinalReport section6.0DATA ANALYSIS Total ug Fluoridein Whole Liver Mean per Dose Group ControlGroup 10 mg/kg dose (T6053) 50 mg/kg dose (T6053) 125 mg/kg dose (T6053) 250 mgakg dose (T6053) ug 15.1 12.7 25.5 17.7 34.7 0()()06r1i RPT1 36L.XLS FC99 PK ID liverspk 63-1 livesrpk 63-2 liverspk 126-1 liverspk 126-2 liverspk 253-1 liverspk 253-2 liverblank-1 F52856-1 F52856-2 F52856-3 F52857-1 F52857-2 F52857-3 F52861 -1 F52861-2 F52861-3 F52862-1 F52862-2 F52862-3 liverblk1 liverbik2 liverspk 1 liverspk 2 F52863-1 F52863-2 F52863-3 SPK 63-1 SPK 63-2 SPK 126-1 SPK 126-2 % rcvry 88% 96% 91% 89% 84% 83% 97% 94% 95% 96% 81% 82% Actual PPM Fin liver (WNV) 1.08 0.970 2.06 2.16 4.16 3.44 0.314 0.250 0.172 0.160 0.150 0.150 0.192 0.186 0.311 0.390 0.237 0.211 0.219 0.412 0.359 1.24 0.857 0.503 0.426 0.395 0.940 0.912 1.76 2.18 Average ppm Fin liver (WNW) 0.194 0.164 0.296 0.222 0.441 liver burned (grams) 0.1224 0.1499 0.1327 0.1242 0.122 0.1462 0.1499 0.136 0.1263 0.1383 0.141 0.1428 0.1038 0.1466. 0.1198 0.1366 0.1456 0.1053 0.136 0.105 0.1157 0.1179 0.1667 0.1199 0.1386 0.1232 0.1525 0.1593 0.1393 0.1136 Whole liver weight (grams) 77.8 77.8 77.8 77.4 77.4 77.4 86.1 86.1 86.1 79.5 79.5 79.5 78.5 78.5 78.5 TotalF- in whole liver (ug) 15.1 12.7 25.5 17.7 34.7 Dosage (mg/kg) 0 10 50 125 250 Page I 9.11.2Summary and raw data;analysisof liverextracts using electrospraymass spectrometry. This data,althoughsupportivei,nthe opinionof the Study Directorisnot requiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. HWI# 6329-136 Study: ProtocolNumber: TestMaterial: Matrix: R Squared Value: RoWnse FactorAmount: Analyst: Date: Method: Instrument: LABOASE File: Single-DoseIntravenouPsharmacokinedc TP8084.PK T-6063InRabbits(FC-99) Liver 0.9816 1.OOE-05 DLC 4/6/95 AMDT-M-4 FisonsVG 2000 ElectrospraMyS 040695A ao&;ao.& A- I 4L*j@4 A --I U/2z195 Group Dose Group 1: 0 mg/kg Group 2: 10 mg /kg Group 3: So mg/kg Group 4: 125 nV/kg Group 5: 250 nig/kg Sampis F52856 IonCount Exbwted wt 9 20514 1.0361 Dilution Concentration Totalmass factor ug/g ofliver 9 1 0.1587 77.8299 Totalamount of FC-95 per liver nV 0.012 F52857 8628 1.0019 1 - F52861 7353 1.1609 1 0.0690 0.0508 77.4061 86.1239 0.005 0.004 F52862 27068 1.0462 1 0.2074 79.5049 0.016 F52863 29919 1.1937 1 0.2009 78.5453 0.016 The concentratiownas calculatebdy usingthestandardcurveand multiplyintgheresultby 4/5.The 4/5 factorIsdu resultofa miscalculatioInnapplyingformula&4 InMethod AMDT-M-4-0. 137 nigoflivewras used In thiscalculatiornatherthanl71 mg. The concentrationIsnthestandardcurvearetherefor5e14largerthanthey shouldbe.By multiplyintghecalculatedconcentratioInnthestandardcurveby 4/5,thecorrectresultIsobtained. 12,1 IaL rFc -14 File:040695A Sample: 040695A ZFS- 6JL169 9 2 5471 LAB-BASE The MS Data Sgstem toltk/9!@@ 41al 06/64/1 15 5 JL5@5 1I9L9484L 5 2J1L335 288 7 33 3 4243 4 4 5345 5 Los- 1024601 13 6 a 774077 33063 2 zFS- 0, ,Son 429612 103 4 877 liOpJL 437 A '\ leag 12506 1515 6/ 3339443J92212L4294499395J5 183223\ 02\298 99 \ 2669 JL.36 2000 30igg 29919 4259 40@ia 2562 534 4284241969 1151,49 5 'BA21K 5000 IY) File:040695A Sample: 040695A I@@- LAB-BASE - The MS Data System 06/04,o 284986 4102 V.FS 169109 36 2 177761 3776 169698 3-921-. 821 3561 1209 3714 210514 36 4 00 Y.FS tL 00 1308 38-91 1134 923 4083 4 ........................ 27068 41105 6 42 41R 8628 06 735 3 00 3779 39 25(Y) U) @b LL .Scn 608 '37'6ai 38 File:040695A Sample:HWI 0406YSA I@@- ZFS- 0' 100- AC-I-XE2@ 4 r-Il LAB-BASE - The MS Data System @2 v A -b A -6")- 10/lq/q-5 CVCX-D 95326 28 lloL95 52 43299 828 4R7 1JL24 371 @@518 163284 219838 87 16IL 4F2937 612 6268325371 Leal 682 857 1JL8SQ1GJ3JL'3L x4.0 220669 a7 06/04,, 15829 11 2 Y.FS.!San 135614 71 70422 2411 L 267 260--- 1897733 339922 JL48497@ 480 5 JL 660 oil@ 429612 1 34 4121 11 I@@@ lag File:040695A Sample:HWI 040695A Los- 74393 Y.FS 25 6 LAB-BASE 112333 26 6 146156 28 8 The MS Data System to/19 q 5 06/04/ 145956 29 8 135711 3148 157098 33l05@ 1284 35 0 I@@ZFS- Son@40 ,F* 1-14 63729 2669 91091 25 8 k- a... - -'26'00 2408 2799 \ x3.1 808 2937 \ 9 1341 3129'-, \@e 117051 28 9 \/J'L8 3 2.23 2 99 9 342495 3149 1043 3288., 391479364737] 6541111 33 6 '444 3,4 06e tA 'i k Zi2'00 39 File:040695A Sample:HWI 040695A .t4S5@6 .too- 56 3 JL ZFS- IL57i JLOO- LAB-BASE t271.t5 5 The MS Data System 5 t365.14 5958 86358 6JL 3 JL586 t742 5732 579.t JL5 9771559 1 58 9 -tg9o 6083- ZFS- JL28625 563.1 s L0 5600 57Qigi .t57255 57@ @8eig 394059 59 a 47948.1 6.t 3 . 60 @a 6.t'oio 06/04/ 86 62 695 6.t5@ 7JL5 62 62,043 A 389722 E ri Method C:DLCLIV Sample Operator Run date 06-23-1995 09:19:00 version: 16 Printed on 06-23-1995 AT 09:19:13 Straight Line Fit forced through origin. --------------------------------- Componen-t#:I + 187049 127117 67071 LEVEL 1 2 3 4 .8 1.6 1.2 AMOUNT Component I EXTERNAL STANDARD AMOUNT AREA ----------- --------- 0.8000 1.2000 67071 127117 1.6000 187049 4.0000 388722 = CALIBRATION 4 LAeeME qLE. 0406q5A y SLOPE X + INTERCEPT --------------------------------------------------- Area Amount = 9.9824E+04 1.001SE-05 Amount Area + O.OOOOE+00 + O.OOOOE+00 R squared 0.9816 9.11.3Summary and raw data; ug F- inwhole liveras determinedby thermalextractiofnollowedby analysisusing Skalarsegmented flow analyzerwith ion selectivelectrode. This data,althoughsupportive,intheopinionof the Study Directorisnotrequiredto reachtheconclusionstatedin FinalReport Section6.0,and thereforeisnot discussedin detail. 00 RE: 6329-136 L IUER SRMPLES RMDT 02495.1 Date of Rnalysis:4-6-95 finalyst:DDW The samples are burned in the Dohrman at 950 C using between 0.1 and 0.2 grams of the liver.The gas is collected in 1.0 mL of 1:1 TiSRB/Milli-Q water then an additional 2 mL of 1:1 TISRB/Milli-Ois added to allow for sufficientvolume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow Rnalyzer using the Ion SpecificElectrode (ISE)Method. TISRB buffer is added to each sample as itproceeds through the system. The sample then goes through a heated mixing coil before the potentialbetween the ion selectiveelectrode and the reference electrode ismeasured. The signal isamplified and related to the fluorideconcentration. The instrument was calibratedin the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride. The standard curve for the high range was plotted using the inverse logarithm option.The standard curve for the low range islinear.Rilstandards and samples were then calculated by the Skalar software using these curves. Hilresults below 0.0001 ppm appear on the raw data as #.####. R quality control standard was analyzed every 10 samples to check for accuracy and drift. Raw data istaken from the appropriate calibratedrange of the Skalar printout and summarized on an Excel spreadsheet. The finalresultsare adjusted for the collectionvolume and any subsequent dilutions. o(JO07() C-A) @-L- . . .. SUMMARY OF 6329-136 LIVERS AMDT 02495.1 GROUP 1 Dose Level :0 GROUP2 Dose Level: 10 mg/kg GROUP3 Dose Level:50 mglkg GROUP4 Dose Level: 125 mgfkg GROUPS Dose Level: 250 mgfkg F52856-1 F52856-2 F52857-1 F52857-2 F52857-3 F52861-1 F52861-2 F52861-3 F52862-1 F52862-2 F52862-3 F52863-1 F52863-2 F52863-3 ND 3.0 0.1360 ND ND 77.8299 0.03 3.0 0.1263 0.68 77.8299 0.08 3.0 0.1410 1.79 77.4061 0.11 3.0 0.1428 2.37 1.39 77.4061 ND 3.0 0.1038 ND 77.4061 ND 3.0 0.1466 ND 86.1239 ND 3.0 0.1198 ND ND 86.1239 0.02 3.0 0.1366 0.49 86.1239 ND 3.0 0.1456 ND 79.5049 ND 3.0 0.1053 ND ND 79.5049 ND 3.0 0.1360 ND 79.5049 0.02 3.0 0.1199 0.62 78.5453 0.03 3.0 0.1386 0.55 0.56 78.5453 0.02 3.0 0.1232 0.52 78.5453 Page 1 1995-07-10 10:23 Operator :DDW OutPutof:950406C I Date oftheAnalysis:1995-04-06 13:33 AnalysiFsileName :C:\SKALAR\DATA\HWIDATA\LIVERS\950406CI 136-LIV.XLS I Tracer 2 Drift 3 Wash 4 Standard 1 5 Standard2 6 Standard3 7 Standard4 8 Standard5 9 Standard6 10 Standard7 11 Standard8 12 Standard9 13 Standard10 14 Drift 15 Wash 16 F52856-1 17 F52856-2 18 BLANK 19 F52857-1 20 F52857-2 21 F52857-3 22 F52861 -1 23 F52861-2 24 F52861-3 25 F52862-1 26 Drift 27 Wash 28 F52862-2 29 F52862-3 30 LIVER BLK I 31 LIVER BLK 2 32 LIVER SPK 1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.49 99% 1.51 101% 0.00 0.015 101% 0.03 99% 0.06 100% 0.09 101% 0.12 99% 0.15 100% 0.29 96% 0.61 102% 1.24 103% 1.46 97% 1.58 105% ND ND 0.03 0.06 0.08 0.11 ND ND ND 0.02 ND 1.56 104% ND ND ND ND ND 0.05 3.0 0.1360 ND 77.8299 ND 3.0 0.1263 0.68 77.8299 53 1.0 0.1499 0.39 3.0 0.1410 1.79 77.4061 139 3.0 0.1428 2.37 77.4061 184 3.0 0.1038 ND 77.4061 ND 3.0 0.1466 ND 86.1239 ND 3.0 0.1198 ND 86.1239 ND 3.0 0.1366 0.49 86.1239 42 3.0 0.1456 ND 79.5049 ND 3.0 0.1053 ND 79.5049 ND 3.0 0.1360 ND 79.5049 ND 3.0 0.1050 ND 3.0 0.1157 ND 3.0 0.1179 1.37 Page 1 0.004 63 136-LIV.XLS 33 LIVER SPK 2 34 F52863-1 35 F52863-2 36 F52863-3 37 SPK 63-1 38 Drift 39 Wash 40 SPK 63-2 41 SPK 126-1 42 SPK 126-2 43 Drift 44 Wash 0.06 0.02 0.03 0.02 0.06 1.50 1.56 104% ND Multi Multi multi 1.50 1.54 103% ND 3.0 0.1667 1.07 3.0 0.1199 0.62 78.5453 49 3.0 0.1386 0.55 78.5453 43 3.0 0.1232 0.52 78.5453 41 3.0 0.1525 1.20 3.0 0.1593 3.0 0.1393 3.0 0.1136 0.004 63 0.004 63 Samples 40-42 had multiplpeeaks duetoinsufficiesnatmpleincup. Disregardresultfsorthesethreesamples.DDW Page 2 '&995-04-06 16:07 OutPut of : 950406Cl Software version 6.1 cl990,93 Operator DDW Date of the Analysis : 1995-04-06 13:33 Analysis File Name : C:\SKALAR\DATA\950406Cl AM%>T Hgom O-L4q S. -J"4.0 Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = 1.##### x - cl Result = 10[ s x = corrected value of the sample cl = corrected value of the concentration I s = Slope of the electrode a2 = al = aO = -0.00000 0.00093 -1.24069 Fluoride L Calibration order = 2 Correlation Result a2 r = 0.99928 x2 + al * x + aO a2 = al = aO = 0.00000 0.00025 0.00292 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height : SAIOOO :1 : 50 sec. : 120 sec. : 1 Sec. : Single : None : 70 mm. Diluter needle Height : 80 rm dilution Factor : 10 dilution Volume : 2.5 ml. Resample :1 Dilution runs 1 User file TXT Reproces No 1995-04-06 16:07 OutPut of Fluoride 1.5 Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off sl standard Ignore s2 standard s3 standard Ignore Ignore s4 standard Ignore s5 standard Ignore s6 standard s7 standard 0.150 0.300 S8 standard S9 standard slO standard Order : Inverse 0.600 1.200 1.500 Logarithm Dimension : PPM start Value trigger Limit Peak shape start ignore eNd ignore Measure window : 500 DU : 1800 Sec : Pointed : 60 Sec : 120 Sec : 75 % Filter Regeneration : No : No formula output Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 : Debubbled : No :0 : No : No : 4095 :0 : Off 950406Cl 0()OOISIL 1995-04-06 16:07 output of 950406Cl sl standard 0.015 s2 standard 0.030 s3 standard 0.060 s4 standard 0.090 s5 standard 0.120 s6 standard 0.150 s7 standard : Ignore s8 standard : Ignore S9 standard : Ignore slO standard : Ignore order : 2 Dimension : PPM start Value : trigger Limit : Peak shape : start ignore : eNd ignore : measure window : 500 DU 1800 Sec Pointed 60 Sec 120 Sec 75 % Filter Regeneration : No : No formula c4:=c3 output Fluoride 1.5 Fluoride L PP)f PP)f Pos Typ Ident Dil Weight wt iw Initial Wash 1 1.000 1t Tracer 1 1.000 2d Drift 1 1.000 Ch Result 3 0.057 4 0.0029 3 1.489 4 1.0431 3 1.508 4 1.0550 Dil F Cor. Valu Time 1 0 280 65 0 0 0 0 1 2284 2571 211 0 2284 0 0 1 2302 2595 387 0 2302 0 0 3w Wash 1 1.000 3 0.057 1 4 0.0029 0 0 300 629 0 0 0 4 sl Standard 1 1 1.000 3 0.064 1 4 0.0152 0 48 348 735 48 0 0 5 s2 Standard 2 1 1.000 3 0.071 4 0.0296 1 102 402 912 0 102 0 0 6 s3 Standard 3 1 1.000 3 0.089 1 211 512 1080 4 0.0601 0 211 0 0 7 s4 Standard 4 1 1.000 3 0.109 4 0.0906 1 313 616 1261 0 313 0 0 8 s5 Standard 5 1 1.000 34 00..1112993 10 440044 7080 14360 9 s6 Standard 6 1 1.000 3 0.154 4 0.1502 1 497 804 1612 0 497 0 0 10 s7 Standard 7 1 1.000 3 0.287 1 863 1178 1786 4 0.2868 0 863 0 0 11 ss Standard 8 1 1.000 3 0.609 1 1387 1716 1962 12 s9 Standard 9 13 slO Standard 10 14 d Drift 15 w Wash 16 u F52856-1 17 u F52856-2 18 u BLANK 19 u F52857-1 20 u F52857-2 21 u F52857-3 22 u F52861-1 23 u F52861-2 24 u F52861-3 25 u F52862-1 26 d Drift 27 w Wash 28 u F52862-2 29 u F52862-3 30 u LIVER BLK 1 31 u LIVER BLK 2 32 u LIVER SPK 1 33 u LIVER SPK 2 4 U.DZJJ 1 1.000 3 1.240 4 0.8957 1 1.000 3 1.459 4 1.0247 1 1.000 3 1.582 4 1.1018 1 1.000 3 0.057 4 0.0029 3 1.000 3 0.189 4 0.0142 3 1.000 3 0.212 4 0.0285 1 1.000 3 0.088 4 0.0586 3 1.000 3 0.313 4 0.0841 3 1.000 3 0.373 4 0.1129 3 1.000 3 0.178 4 0.0070 3 1.000 3 0.178 4 0.0067 3 1.000 3 0.189 4 0.0142 3 1.000 3 0.201 4 0.0221 3 1.000 3 0.186 4 0.0119 1 1.000 3 1.555 4 1.0843 1 1.000 3 0.057 4 0.0029 3 1.000 3 0.183 4 0.0103 3 1.000 3 0.183 4 0.0098 3 1.000 3 0.185 4 0.0113 3 1.000 3 0.181 4 0.0090 3 1.000 3 0.256 4 0.0540 3 1.000 3 0.265 u u 1 2053 2402 2136 0 2053 0 0 1 2256 2616 2312 0 2256 0 0 1 2372 2668 2485 0 2372 0 0 1 0 296 2717 0 0 0 0 3 44 342 2834 0 44 0 0 3 98 400 3009 0 98 0 0 1 206 512 3181 0 206 0 0 3 292 600 3360 0 292 0 0 3 384 696 3536 0 384 0 0 3 16 320 3676 0 16 0 0 3 15 320 3869 0 15 0 0 3 44 352 4053 0 44 0 0 3 74 384 4235 0 74 0 0 3 35 345 4411 0 35 0 0 1 2346 2657 4587 0 2346 0 0 1 0 312 4751 0 0 0 0 3 29 340 4934 0 29 0 0 3 27 336 5104 0 27 0 0 3 33 340 5286 0 33 0 0 3 24 329 5461 0 24 0 0 3 190 496 5637 0 190 0 0 3 208 512 5807 04 u F52863-1 35 u F52863-2 36 u P52863-3 37 u SPK 63-1 38 d Drift 39 w Wash 40 u SPK 63-2 41 u SPK 126-1 42 u SPK 126-2 43 d Drift 44 w Wash wt rw RunOut Wash 4 U.VDVZ 3 1.000 3 0.206 4 0.0247 3 1.000 3 0.207 4 0.0253 3 1.000 3 0.201 4 0.0215 3 1.000 3 0.269 4 0.0612 1 1.000 3 1.563 4 1.0896 1 1.000 3 0.057 4 0.0029 3 1.000 3 Multi 4 0.3527 3 1.000 3 Multi 4 0.3518 3 1.000 3 Multi 4 0.3406 1 1.000 3 1.544 4 1.0776 1 1.000 3 0.057 4 0.0029 1 1.000 3 0.057 4 0.0029 u zvo v v 3 84 384 5985 0 84 0 0 3 86 384 6153 0 86 0 0 3 72 368 6336 0 72 0 0 3 215 512 6510 0 215 0 0 1 2354 2644 6688 0 2354 0 0 1 0 288 6861 0 0 0 0 3 M 1020 1360 7103 0 1020 0 0 3 M 1018 1388 7160 0 1018 0 0 3 M 992 1392 7344 0 992 0 0 1 2336 2752 7558 0 2336 0 0 1 0 448 7743 0 0 0 0 1 0 1405 8033 0 0 0 0 00()O,Sll 1.9832492i Calibration curve of 9SO406CI Fluoride I.S o-oIS8t89im ir4tv.614 0 c ol 0.05745[)3-@@ 0 Order Measured Inverse Logarithm 409-ci S 3ii-9 2.Sc@-178708 Calibration curve of 950406CI Fluoride L ().()C)44394=i--317MP4 10 p4 c 0.0029"32 0 Order 2 Measured 409S r 0.89928 Raw data of 95040SCI : Fluoride 1.5 m 0 MU-rRr-M 28o -6,4 4095 OL6 11 ILO 0 0 Esc=Exit 40,95 T ime Fl=iielp Crtl-P=Edit Peaks Raw data of 350406Cl : Fluoride 1.5 3860 nkfimm 3o8 [MiPWr,-47M 5000 10 38C'o Esc=Exit FI=Help TimpCrti-P=Edit peaks 8860